Project Summary Abstract. NUT midline carcinoma (NMC), with a median survival of 6.7 months, is one of the most aggressive solid tumors known. It is a subtype of squamous cell carcinoma characterized by translocation of the NUT (aka NUTM1) gene, most commonly forming a fusion to the double-bromodomain encoding protein (BET), BRD4. There is an urgent need for the identification of more specific therapeutic targets in NMC. The over-reaching goal of this proposed project is to greater understand the mechanism of BRD4-NUT oncogenesis and identify effective therapeutic targets for treating this disease. BRD4-NUT functions to block differentiation and maintain proliferation of NMC cells, largely through activation of MYC expression. This function is disrupted upon treatment with BET inhibitors, which as acetyl- lysine mimetics prevent binding of BRD4 bromodomains to acetylated chromatin. BRD4-NUT drives the expression of pro-growth target genes, including MYC, through the formation of megabase-sized massive hyperacetylated 'megadomains'. BRD4-NUT megadomains arise from pre-existing active enhancers and spread to fill cell-type-specific topologically associating domains (TADs). TADs are higher order genomic structures whose function is to orchestrate cell-fate determining transcriptional programs through DNA-DNA contacts. Unique proteins recruited by BRD4-NUT recently identified by our group include the histone acetyl- transferase (HAT), p300, and several ZNF proteins collectively termed Z4. These findings indicate that BRD4- NUT ?hijacks? cell-type specific TADs to drive transcription of pro-growth, anti-differentiative genes as postulated in the following model: First, BRD4-NUT complex proteins seed regions corresponding to cell-type- specific active TADs through the chromatin-binding of BRD4. Second, megadomains form from contiguous expansion of BRD4-NUT complexes across chromatin in a feed-forward manner dependent upon p300 HAT activity. Third, megadomain size is limited by TAD boundaries and HDAC activity recruited by the Z4 complex. Fourth, hyperacetylated chromatin, recruitment of chromatin remodelers, and upregulation of cis lncRNAs changes the chromatin configuration to enhance DNA-DNA interactions to drive transcription of key pro- growth, anti-differentiative genes. The goals of this proposal are to test this hypothetical model, as listed in the specific aims below. Aim 1. To determine how BRD4-NUT megadomains form. Aim 2. To determine how BRD4-NUT megadomains function. Impact. Successful completion of the aims is expected to identify key BRD4-NUT-associated proteins in pathologic megadomain formation, and will identify novel and possibly more effective therapeutic targets in NMC and other cancers. In addition, we predict that BRD4-NUT megadomains will provide a model with far- reaching impact on the structure-function relationship of chromatin conformation in cancer and development.